PCR ribotyping and arbitrarily primed PCR for the comparison of enterotoxigenic Bacteroides fragilis strains from two Polish university hospitals

Objective: To study the clinical incidence and possible clonal relatedness of enterotoxigenic strains of Bacteroides fragilis among pediatric and adult patients in two Polish university hospitals.
Method: Fecal samples from 201 adults and 131 infants (with or without diarrhea) and vaginal samples from 100 pregnant women nursed in two Polish university hospitals were analyzed with respect to carriage of enterotoxin-producing Bacteroides fragilis (ETBF). This putative pathogen was identified by cultivation and subsequent cytopathogenicity testing of culture supernatants on HT/29 C1 cells.
Results and discussion: Two ETBF strains were isolated from childrens' feces; two additional strains were isolated from adults, and from the vaginal samples only a single strain was isolated. One strain (W2) was isolated from a child with diarrhea. These incidence figures, the fact that all ETBF isolates were shown to produce strongly differing amounts of the cytotoxin, and the genetic unrelatedness of the strains as demonstrated by two different PCR-mediated DNA typing procedures, indicates that clonal spread of ETBF is presently not a clinical problem in these hospitals. It was shown that PCR-mediated ribotyping and arbitrarily primed PCR can be applied with success to study the epidemiology of ETBF.     

Detection of enterotoxigenic Bacteroides fragilis by PCR.

Strains of enterotoxigenic Bacteroides fragilis (ETBF) are associated with diarrhea in young farm animals and, at least in particular settings, in children. Enterotoxin production by ETBF is currently detected by a tissue culture assay with HT-29 cells. We have developed a PCR assay based on the detection of the enterotoxin gene to identify ETBF in culture and in stool samples. Overall, 113 bacterial strains were examined, including 3 B. fragilis reference strains, 75 B. fragilis isolates (comprising 40 ETBF isolates), 20 Bacteroides spp. other than B. fragilis, and 15 strains belonging to other genera. Complete agreement was found between the results of the tissue culture assay and those of the PCR for our strains. PCR was also used to detect ETBF directly in fecal samples. Stools from two healthy volunteers were spiked with known numbers of ETBF and were processed by three different methods. A culture method, which required inoculation of the stools on selective plates and the collection of the whole bacterial growth ("sweeps"), was found to be the most sensitive. PCR performed with the plate sweeps yielded amplification products with a detection limit of 10(5) to 10(4) CFU/g of feces. By this method 18 samples of diarrheic stools (10 positive and 8 negative for ETBF) were examined. The results of the PCR were in accordance with the culture results in all cases. The proposed PCR assay represents a diagnostic tool for the rapid identification of ETBF in culture as well as in fecal samples.     

Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86-5433-2-2).     

Identification of a Third Metalloprotease Toxin Gene in Extraintestinal Isolates of Bacteroides fragilis

To further understand the epidemiology of enterotoxigenic Bacteroides fragilis (ETBF), 89 extraintestinal B. fragilis strains from Seoul, Korea, were examined for secretion of B. fragilis toxin (BFT) by the HT29/C1 biologic assay and for the B. fragilis toxin gene (bft) by colony blot hybridization and PCR. Complete agreement between the three techniques was found. Overall, 34 B. fragilis strains (38%) were identified as ETBF. Eleven of the 34 ETBF strains (32%) expressed a new isoform of BFT (Korea-BFT). This new isoform is more related to BFT-2 than to BFT-1. Like BFT-1 and BFT-2, Korea-BFT cleaves E-cadherin, the zonula adherens protein.     

Diversity of the metalloprotease toxin produced by enterotoxigenic Bacteroides fragilis

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a 20-kDa zinc metalloprotease toxin (BFT) associated with diarrheal disease of animals, young children, and adults. BFT stimulates secretion in intestinal loops in vivo and modifies epithelial cell morphology in vitro. The B. fragilis toxin (bft) gene from ETBF strain 86-5443-2-2 (piglet; bft-2) revealed significant nucleotide and predicted amino acid differences when compared to the bft gene from ETBF strain VPI 13784 (lamb; bft-1). This study compares BFT-1 and BFT-2, respectively, produced by ETBF strains VPI 13784 and 86-5443-2-2 purified using the Van Tassell method (38) and a modified purification scheme described herein. Multiple differences in the protein toxins produced by these ETBF strains were identified. First, purified BFT-1 eluted from a high-resolution anion-exchange column (Mono Q) at 0.22 +/- 0.005 M NaC1 versus 0.18 +/- 0.001 M NaC1 for BFT-2 (P < 0.001). Second, BFT-1 and BFT-2 exhibited different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase fast protein liquid chromatography. Third, each BFT reacted with greater specificity to homologous rather than heterologous antisera. Fourth, BFT-2 had modest, but consistently, greater biological activity than BFT-1 when tested on HT29/C1 cells (P < or = 0.01). Together, these data indicate that these ETBF strains produce two distinct isotypes of BFT, termed BFT-1 (VPI 13784 BFT) and BFT-2 (86-5443-2-2 BFT) to recognize the order in which the proteins were purified and genetic sequences identified. The modified purification scheme described in this report yields about two to three times more purified BFT protein than previous protocols and is less time consuming.     

The alleles of the bft gene are distributed differently among enterotoxigenic Bacteroides fragilis strains from human sources and can be present in double copies

Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms bft-1 and bft-2 or bft-3 borne by ETBF. By sequencing the portion of the bft gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as bft-2 or bft-3, we found in our collection two strains harboring bft-3, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that bft-1 is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while bft-2 is present in isolates from feces of children. This association is statistically significant (P, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the bft and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring bft-1 (three strains) or bft-2 (one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring bft-3, either in a single copy or in double copies, produced the smallest amount of toxin in our collection.     

Application of the French guidelines for preventing neonatal group B streptococcal disease in a university hospital

This study evaluated the application of the French guidelines for prevention of neonatal group B streptococcus (GBS) infections. The prevalence of GBS vaginal carriage by pregnant women during the study period was 6%. Less than 50% of pregnant women testing positive for GBS were treated with at least two doses of antibiotics during labour, and most received only one dose or no antibiotics. In addition, several neonates were colonised or infected by GBS although their mothers were GBS-negative. These results are consistent with vaginal screening having a poor sensitivity, as suggested by the low prevalence of GBS carriage.     

Possible role of enterotoxigenic Bacteroides fragilis in the etiology of infectious vaginitis

Vaginitis is a common gynecologic disorder. It is due to several causes, some even unknown. Bacteroides fragilis is the most important anaerobe in clinical bacteriology, some strains of this group are notable for being enterotoxigenic and they have been associated with intestinal and extraintestinal syndromes. They have recently been isolated from patients with vaginitis. The purpose of this study was to investigate a possible association of enterotoxigenic B. fragilis with infectious vaginitis. 265 samples of vaginal exudate were processed, 202 from symptomatic patients and 63 healthy women. The identification of the microorganisms was carried out by conventional methods. In 31.2% of symptomatic patients were identified: Gardnerella vaginalis, Mobiluncus, Candida albicans, Mycoplasma hominis, Ureaplasma urealyticum and Streptococcus agalactiae. B. fragilis was identified in 27 symptomatic patients and 5 healthy women. These strains were cultivated in liquid medium and incubated during 48 h at 36 degrees C in anaerobe chambers. Supernatant activity was assayed in HT-29 cells. Eighteen B. fragilis strains isolated from symptomatic patients were enterotoxigenic, because induced alterations in target cell morphology. It was not identified in healthy women (P < 0.05). 77.7% of enterotoxigenic B. fragilis strains were not associated with other specific pathogens. This fact suggests that enterotoxigenic B. fragilis could be a cause for vaginitis. The effect of enterotoxin on E-cadherin of vaginal epithelium could facilitate invasion and its possible pathogenic role in the vagina. This is the first report that associates enterotoxigenic Bacteroides fragilis as a possible cause of infectious vaginitis.     

Genetic dissimilarity of commensal strains of Candida spp. carried in different anatomical locations of the same healthy women.

Candida spp. carriage and strain relatedness were assessed in 52 healthy women at 17 anatomical locations by using an isolation procedure which assesses carriage intensity and by using a computer-assisted DNA fingerprinting system which computes genetic similarity between strains on the basis of the patterns of Southern blots probed with the moderately repetitive sequence Ca3. Candida spp. were cultured from 73% of the test individuals, most frequently from the oral (56%), vulvovaginal (40%), and anorectal (24%) regions. Half of the test individuals with Candida spp. carried the organism simultaneously in more than one of the three general areas of carriage. Isolates from different body locations of the same individual were either completely unrelated, identical, or highly similar but nonidentical. In 11 cases in which Candida spp. were simultaneously isolated from the oral cavity and vaginal canal, seven pairs of isolates were genetically unrelated and four pairs were similar but nonidentical. In the latter cases, the isolate pairs each appear to have arisen by genetic divergence from a single progenitor. A comparison of the genetic relatedness of isolates from different individuals further uncovered a single strain which was vaginospecific in the Iowa City, Iowa area and reduced genetic diversity among vulvovaginal strains compared with those isolated from other body locations. These results suggest that strains adapt to different anatomical locations and, conversely, that in a healthy individual there is anatomical selection of vaginotropic, anotropic and orotropic strains of Candida spp.     

A possible role of Bacteroides fragilis enterotoxin in the aetiology of colorectal cancer

The prevalence of enterotoxigenic Bacteroides fragilis (ETBF) was investigated in stool specimens from 73 patients with colorectal cancer and from 59 control patients. Stool specimens were cultured on Bacteroides Bile Esculin agar and B. fragilis was identified by conventional methods. After DNA extraction, the enterotoxin gene (bft) was detected by PCR in 38% of the isolates from colorectal cancer patients, compared with 12% of the isolates from the control group (p 0.009). This is the first study demonstrating an increased prevalence of ETBF in colorectal cancer patients.     

Rapid detection of group B streptococci in pregnant women at delivery

BACKGROUND: Group B streptococcal infections are an important cause of neonatal morbidity and mortality. A rapid method for the detection of this organism in pregnant women at the time of delivery is needed to allow early treatment of neonates. METHODS: We studied the efficacy of two polymerase-chain-reaction (PCR) assays for routine screening of pregnant women for group B streptococci at the time of delivery. We obtained anal, vaginal, and combined vaginal and anal specimens from 112 pregnant women; in 57 women, specimens were obtained before and after the rupture of the amniotic membranes. The specimens were tested for group B streptococci by culture in a standard selective broth medium, with a conventional PCR assay, and with a new fluorogenic PCR assay. RESULTS: Among the 112 women, the results of the culture of the combined vaginal and anal specimens were positive for group B streptococci in 33 women (29.5 percent). The two PCR assays detected group B streptococcal colonization in specimens from 32 of these 33 women: the one negative PCR result was in a sample obtained after the rupture of membranes. As compared with the culture results, the sensitivity of both PCR assays was 97.0 percent and the negative predictive value was 98.8 percent. Both the specificity and the positive predictive value of the two PCR assays were 100 percent. The length of time required to obtain results was 30 to 45 minutes for the new PCR assay, 100 minutes for the conventional PCR assay, and at least 36 hours for culture. CONCLUSIONS: Colonization with group B streptococci can be identified rapidly and reliably by a PCR assay in pregnant women in labor both before and after the rupture of membranes.     

围产期孕妇生殖道B族链球菌感染和耐药情况及其对妊娠结局的影响

目的 分析围产期孕妇生殖道B族链球菌(GBS)感染情况以及对妊娠结局的影响.方法 收集2014年1月至2017年1月期间在我院进行产前检查的476例35～ 37周孕妇生殖道分泌物,通过实时定量PCR技术以及培养法检测B族链球菌感染情况,对分离培养出的GBS菌株进行药敏试验,并将实时定量PCR检测GBS阳性孕妇按不同治疗方案分为A、B两组,A组孕妇分娩开始即静脉注射敏感性抗生素直至分娩结束,B组孕妇在发现GBS阳性时即口服抗生素7d,其余治疗方法同A组,分析两组治疗方案孕妇妊娠结局.结果 476例孕妇围产期生殖道分泌物经实时定量PCR检测GBS阳性者148例,阳性率为31.1％,细菌培养法检测阳性者38例,阳性率为8.0％.分离培养的38株GBS对青霉素、万古霉素、利奈唑胺、红霉素、左氧氟沙星、克林霉素的耐药率为0％、0％、0％、23.7％、31.6％、52.6％.A组孕妇宫内感染、胎膜早破、早产以及新生儿感染发生率均高于B组孕妇,两组差异有统计学意义(P＜0.05).结论 围产期孕妇生殖道GBS感染发生率高,GBS感染可导致宫内感染、胎膜早破、早产以及新生儿感染等不良妊娠结局发生率增加,GBS感染后采用敏感性抗生素预防治疗可以改善孕妇妊娠结局.     

Rapid and Sensitive Assay for Detection of Enterotoxigenic Bacteroides fragilis

Bacteroides fragilis is an obligatory anaerobic, gram-negative bacterium found among the normal intestinal flora of humans. Enterotoxigenic strains of B. fragilis (ETBF) have been associated with diarrheal diseases in humans and animals. The enterotoxin of ETBF induces fluid changes in ligated intestinal segments and cytotoxic response in HT29/C1 cells. By using a pair of monoclonal antibodies (MAbs; MAb C3 and MAb 4H8) specific for the lipopolysaccharide of B. fragilis, an assay based on immunomagnetic separation (IMS) in combination with PCR (IMS-PCR) was developed. After DNA extraction, a 294-bp fragment was amplified. The specificity of the IMS-PCR assay was evaluated by adding previously isolated and confirmed ETBF strains to normal fecal samples. All fecal samples to which ETBF strains were added were positive, showing a 100% specificity. The spiked fecal samples were also used for evaluation of the sensitivity of the assay. The detection limit was found to be ~50 CFU/g of feces. By this method 10 clinical fecal samples (5 from patients with diarrhea and 5 from healthy controls) were examined. The results of PCR were in accordance with the results of the HT29/C1 cell assay for all samples. The minimum time to retrieval of the final result by the IMS-PCR method is 36 h. The proposed IMS-PCR assay is rapid and sensitive for the direct detection of ETBF in stool samples.     

Species and genotypic diversities and similarities of pathogenic yeasts colonizing women

We examined the patterns of strain relatedness among pathogenic yeasts from within and among groups of women to determine whether there were significant associations between genotype and host condition or body site. A total of 80 yeast strains were isolated, identified, and genotyped from 49 female volunteers, who were placed in three groups: (i) 19 women with AIDS, (ii) 11 pregnant women without human immunodeficiency virus (HIV) infection, and (iii) 19 women who were neither pregnant nor infected with HIV. Seven yeast species were recovered, including 59 isolates of Candida albicans, 9 isolates of Candida parapsilosis, 5 isolates of Candida krusei, 3 isolates of Candida glabrata, 2 isolates of Saccharomyces cerevisiae, and 1 isolate each of Candida tropicalis and Candida lusitaniae. Seventy unique genotypes were identified by PCR fingerprinting with the M13 core sequence and by random amplified polymorphic DNA analysis. Of the nine shared genotypes, isolates from three different hosts were of one genotype and pairs of strains from different body sites of the same host shared each of the other eight genotypes. Genetic similarities among groups of strains were calculated and compared. We found no significant difference in the patterns of relatedness of strains from the three body sites (oral cavity, vagina, and rectum), regardless of host conditions. The yeast microflora of all three host groups had similar species and genotypic diversities. Furthermore, a single host can be colonized with multiple species or multiple genotypes of the same species at the same or different body sites, indicating dynamic processes of yeast colonization on women.     

Bilan bactériologique de six ans de dépistage du streptocoque de groupe B (SGB) au cours du dernier trimestre de la grossesse

Streptococcus agalactiae (GBS) is a significant cause of morbidity and mortality among newborns. Colonization frequently occurs in pregnant women, nearly all international recommendations suggest that all pregnant women must be screened for vaginal colonization at 34 to 37 weeks of gestation. The microbiological diagnostic modalities used to combat GBS had to be accurate and in short time frame. We reported a 6 years experience of GBS screening, comparing results of culture swab of prenatal vaginal specimens and newborns colonization or infection. The carriage rate of 13 to 14% of GBS in newborn was unchanged during all the study period.     

妊娠35～37周孕妇携带的B群无乳链球菌血清分型

目的 探讨北京地区妊娠35～37周孕妇携带的无乳链球菌(GBS)血清型的分布特点.方法 按照2010年美国CDC颁布的指南中的推荐对6000名妊娠35 ～ 37周的孕妇进行GBS常规筛查.阴道和直肠分泌物联合取样进行分离培养,用VITEK 2 Compact全自动细菌鉴定系统对疑似B群链球菌进行鉴定,使用多重PCR扩增及基因测序的方法对GBS菌株进行血清分型检测.结果 共检出311株无乳链球菌,分为8个血清型.血清分型为Ⅰa型106例(34.1％),Ⅰb型53例(17.4％),Ⅲ型134例(43.1％),Ⅳ型6例(2.3％),Ⅱ型5例(1.6％),Ⅷ型3例(0.96％),Ⅴ型2例(0.64％),Ⅸ型2例(0.64％).结论 北京地区妊娠35 ～ 37周孕妇携带的无乳链球菌血清分型主要为Ⅲ、Ⅰa型.     

Rapid screening for Streptococcus agalactiae in vaginal specimens of pregnant women by fluorescent in situ hybridization

Group B streptococci (GBS) are the most frequent pathogens in neonates with sepsis. A rapid screening method is required to identify carriage of GBS in pregnant women at the time of delivery. In order to detect GBS in vaginal specimens, the efficiency of the standard culture versus fluorescent in situ hybridization (FISH) was investigated. In 258 examined vaginal specimens, FISH identified 58 of the 59 GBS-positive samples (98.3%), whereas by means of standard culture only 38 specimens were positive (64.4%). We recommend FISH as a rapid, specific, highly sensitive screening technique for the detection of GBS in pregnant women at delivery.     

Group B streptococci in women fitted with intrauterine devices.

A survey was made of group B streptococcal carriage at various sites in 100 women attending a clinic for the insertion of an intrauterine contraceptive device (IUD). Twenty-three women carried streptococci at one or more sites at the preinsertion visit, the vaginal carriage rate being 16%. Six months after insertion changes in carrier status were noted and there was evidence of a change of strain in four patients. Twenty-nine women were carriers at one or more sites at some stage of the study. There was no evidence that symptoms attributable to infection in patients fitted with an IUD were caused by group B streptococci.     

Bacteroides fragilis enterotoxin induces intestinal epithelial cell secretion of interleukin-8 through mitogen-activated protein kinases and a tyrosine kinase-regulated nuclear factor-kappaB pathway

Enterotoxigenic Bacteroides fragilis (ETBF) secretes a 20-kDa metalloprotease toxin termed B. fragilis toxin (BFT). ETBF disease in animals is associated with an acute inflammatory response in the intestinal mucosa, and lethal hemorrhagic colitis may occur in rabbits. In this study, we confirmed recent reports (J. M. Kim, Y. K. Oh, Y. J. Kim, H. B. Oh, and Y. J. Cho, Clin. Exp. Immunol. 123:421-427, 2001; L. Sanfilippo, C. K. Li, R. Seth, T. J. Balwin, M. J. Menozzi, and Y. R. Mahida, Clin. Exp. Immunol. 119:456-463, 2000) that purified BFT stimulates interleukin-8 (IL-8) secretion by human intestinal epithelial cells (HT29/C1 cells) and demonstrate that stimulation of IL-8 production is dependent on biologically active BFT and independent of serum. Induction of IL-8 mRNA expression occurs rapidly and ceases by 6 h after BFT treatment, whereas IL-8 secretion continues to increase for at least 18 h. Our data suggest that BFT-stimulated IL-8 secretion involves tyrosine kinase-dependent activation of nuclear factor-kappaB (NF-kappaB) as well as activation of the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-related kinase. Simultaneous activation of NF-kappaB and MAPKs appears necessary for secretion of IL-8 by HT29/C1 cells treated with BFT.     

Detection of imipenem-resistant and metronidazole-resistant Bacteroides fragilis group strains in fecal samples

Objective: To investigate the imipenem and metronidazole resistance profiles of Bacteroides fragilis group strains in fecal samples and to detect the resistance genes ( ccrA and nim ) coding for imipenem and metronidazole resistance in B. fragilis group strains.
Methods: In total, 925 fecal samples, 729 from consecutive diarrhea patients and 196 from healthy controls, were collected at Huddinge University Hospital in 1997. A modified disk diffusion method was employed to screen for imipenem-resistant and metronidazole-resistant B. fragilis group strains. In strains considered resistant by the modified disk diffusion method, the minimum inhibitory concentrations (MICs) were further determined by the agar dilution method. PCR assays were used to detect the carbapenem-hydrolyzing metallo-β-lactamase gene ( ccrA ) and the 5-nitroimidazole resistance genes ( nim ) in pure cultures (purePCR), directly from fecal samples through direct broth enrichment (dirPCR) and by immunomagnetic separation (imsPCR).
Results: Two imipenem-resistant B. fragilis strains, one of which was simultaneously resistant to metronidazole, and two B. fragilis group strains with MICs near the breakpoint for metronidazole resistance, were isolated from the fecal samples of diarrhea patients. The ccrA gene was identified in all the imipenem-resistant B. fragilis strains by purePCR, dirPCR and imsPCR. The nim genes were also detectable by these PCR assays.
Conclusions: The incidences of imipenem-resistant and metronidazole-resistant B. fragilis group strains were low in the investigated diarrhea patients. Simultaneous resistance to imipenem and metronidazole is of great concern in clinical medicine, and the proposed PCR assays may be useful in epidemiologic studies of distribution of resistance genes in the fecal microflora.